We have explored molecular details involved in the up-regulation of delta opioid receptors in hybrid neuroblastoma NG 108-15 cells treated with the opioid antagonist naltrexone for two days. The up-regulation is associated with an increase in 3H-DADLE and 3H-diprenorphine Bmax values in both light and heavy membrane fractions derived from subcellular fractionations. In contrast, a 5-min exposure to the opioid antagonist naltrexone or ICI 174864 induces a transient down-regulation of delta-opioid receptors prior to up-regulation. Naltrexone and delta-specific antagonists ICI 174864 and naltrindole also diminish specific activities of the lysosomal enzymes beta-glucuronidase and beta-hexoseaminidase. Pretreatment of cells with concanavalin A blocks both the down-regulation and alterations in the lysosomal enzyme activities, suggesting that the initial process of up-regulation of the delta-opioid receptors by antagonists entails down-regulation that may involve lysosomal enzyme activity changes. We also have identified novel intracellular delta-opioid binding sites associated with the nuclei of the same cell line. These nuclear delta-opioid sites have been determined by immunohistochemical studies on cryostat sections with an anti-opioid receptor antibody and by the Kd and Bmax values of the binding of 3H-diprenorphine to the highly purified nuclear preparation. Opioid binding sites have also been shown in subnuclear preparations. Agonists 3H-DADLE and 3H-DSLET bind with high affinity to nuclear membranes and with lower affinity to chromatin. In contrast, the partial agonist 3H-diprenorphine high affinity binding sites are predominant in chromatin, while low affinity sites are in the nuclear membrane. Accordingly, GppNHp sensitivity of 3H-DADLE binding is detected in nuclear membranes but not in chromatin. Both agonist and partial agonist binding sites in nuclear membrane and chromatin are abolished by treatment of cells with cycloheximide. Taken together the results sug- gest that NG 108-15 cells contain newly synthesized G protein-coupled delta-opioid receptors in nuclear membrane and internalized, uncoupled opioid binding sites in chromatin.